Karyotypes of six spider species belonging to the families Gnaphosidae, Salticidae, Thomisidae, and Zodariidae (Araneae) from Turkey

Abstract In this study, the karyotypes of six spider species from Turkey belonging to the families Gnaphosidae, Salticidae, Thomisidae, and Zodariidae were analyzed. Male chromosomal features including diploid chromosome numbers and sex chromosome systems were determined as 2n=22, X1X20 in Drassyllus sur Tuneva & Esyunin, 2003, Nomisia exornata (C. L. Koch, 1839), and Nomisia orientalis Dalmas, 1921; 2n=28, X1X20 in Sitticus caricis (Westring, 1861); 2n=23, X0 in Xysticus gallicus Simon, 1875 and 2n=42, X1X20 in Pax islamita (Simon, 1873), respectively. The chromosome morphology of all species was acrocentric. Data obtained contribute to knowledge of the karyotype evolution of araneomorphs.


Introduction
Spiders are one of the most important animal groups, and contain approximately 44 500 species all around the world (Platnick 2014) and consists of three primary clades, namely Mesothelae, Mygalomorphae and Araneaomorphae, the last one being phylogenetically most derived and the largest group (Coddington and Levi 1991). Infraorder Araneaomorphae contains more than 41 000 species (Platnick 2014). Despite this diversity, only 771 species of spiders have been karyotyped so far (Araújo et al. 2014).
Entelegyne spiders form a very diversified clade of araneomorphs. Their karyotypes are characterized by a predominance of acrocentric chromosomes, X 1 X 2 0 sex chromosome system (Araújo et al. 2005), relatively low diploid chromosome numbers (ranges from 10 to 49, Kořínková and Král 2013), and chiasmatic meiosis (Kumbıçak 2010). Acrocentric karyotypes of entelegynes with lower chromosome numbers could be derived from ancestral acrocentric karyotypes by tandem fusions (Suzuki 1954) or by cycles of centric fusions and subsequent pericentric inversions . The latter hypothesis is supported by the fact that centric fusions are the most frequent source of chromosome polymorphism found in populations of entelegyne spiders .
In spiders, the X 1 X 2 0 system could be the ancestral sex chromosome determination as inferred from its presence in the most primitive recent spiders, namely the suborder Mesothelae and basal families of the infraorder Mygalomorphae (Suzuki 1954).
Salticidae, Thomisidae, Gnaphosidae, and Zodariidae are some of the largest families in the order Araneae (Platnick 2014). Despite the high diversity of these spider groups, cytogenetic data have been collected only in 223 species belonging to these clades (Araújo et al. 2014).

Material and methods
Material: Spiders were collected in Mediterranean, Southeast and Central Anatolia (Turkey) during the year 2012. Collection data of particular species (localities including their coordinates, dates of collection, number of individuals studied) are listed in Table 1. Voucher specimens were deposited in the collection of Department of Molecular Biology and Genetics, Art and Science Faculty, Nevşehir Hacı Bektaş Veli University (Nevşehir, Turkey). The identification of spiders was made by O. Seyyar (Department of Biology, Art and Science Faculty, Niğde University, Niğde, Turkey).
Chromosome preparations and observation: Slides for chromosome observations were made by the spreading technique of Traut (1976), with some modifications. This method consisted of three basic steps. First, the gonads were hypotonized in 0.075 M KCl for 12-15 min in room temperature (RT). Second, gonads were fixed in two batches of freshly prepared Carnoy fixative (ethanol: chloroform: glacial acetic acid; 6:3:1), first batch for 10 min and second one for 20 min (RT). Finally, a cell suspension was prepared from a piece of tissue in a drop of 60% acetic acid on a slide using a pair of tungsten needles. The slide was placed on a histological plate at 42 °C and the drop was evaporated by mov-ing it with a tungsten needle. Slides were stained with 5% Giemsa in Sørensen phosphate buffer (pH=6.8) for 27 min (RT). Chromosome spreads were investigated an Olympus BX53 microscope and photographed using a DP26 digital camera (Olympus) using CELLSENS software (Olympus). Relative chromosome lengths (RCL) including standard deviations were calculated as a percentage of the total chromosome length of the diploid set including sex chromosomes (%TCL) from 10 mitotic metaphase plates for each species by CELLSENS software. Classification of chromosome morphology was based on the arm ratio (Levan et al. 1964).
The sex chromosomes were positively heteropycnotic from leptotene to diakinesis in both Nomisia species studied. Plates consisted of 10 autosomal bivalents and two univalent sex chromosomes from pachytene to metaphase I (Fig. 2C-E). At meiotic anaphases, 10 chromosomes segregated to one pole and 12 chromosomes to another pole (Fig. 2F).
Leptotene, zygotene, and pachytene nuclei included a positively heteropycnotic sex chromosome body that was located at the periphery of the nucleus (Fig. 2G). At late prophase I (i.e. diplotene and diakinesis), 13 autosomal bivalents and two univalent sex chromosomes were determined (Fig. 2H).
From beginning of pachytene to metaphase I, plates consisted of 20 autosomal bivalents and two not associated sex chromosomes on the periphery of nucleus (Fig.  2K). Sex chromosomes were positively heteropycnotic during prophase and metaphase II. Metaphases II consisted of 20 or 22 chromosomes, respectively. Metaphases II with 22 chromosomes contained two X chromosomes (Fig. 2L).

Discussion
Karyotypes of 771 spider species from 277 genera are known at present (Araújo et al. 2014). Diploid chromosome numbers of spiders range from 7 (Suzuki 1954) to 128 . Entelegynae araneomorphs exhibit lower diploid numbers and mostly monoarmed chromosomes when compared with the predominantly high chromosome numbers and biarmed chromosomes of mygalomorphs . The sex chromosome system of most entelegynes is X 1 X 2 0 type. This system is supposed to be the ancestral form in spiders. It was found in more than 77% of spiders (Araújo et al. 2005).
The male karyotype of Xysticus gallicus displays the general pattern described for most Thomisidae: a diploid chromosome number 23 and X0 sex chromosome system including acrocentric sex chromosome. All Xysticus species analyzed so far present this karyotype with exception of X. triguttatus Keyserling 1880 (Painter 1914). According to the phylogeny of Benjamin et al. (2008), Xysticus is sister group to Coriarachne Thorell, 1870, that also presents 2n=23, X0 (C. fulvipes Karsch, 1879;Suzuki 1952). There are two hypotheses explaining the origin of acrocentric X0 sex chromosome system in spiders. According to Chatterjee (1989, 1992) the acrocentric X chromosome can be derived by centric fusion of the X 1 and X 2 chromosomes, followed by pericentric inversions. Also, the acrocentric X chromosome could have originated from tandem fusion between acrocentric X 1 and X 2 chromosomes (Pekár and Král 2001).
Our study represented a diploid number of 42 acrocentric chromosomes and X 1 X 2 0 system in Pax islamita. This finding is compatible with the results reported by Král et al. (2011). However, these authors have also found heterozygotes for autosomal centric fusion (2n♂=41, X 1 X 2 0) in addition to the standard individuals. Furthermore, they revealed different pattern of sex chromosome behaviour at male germline of this species. According to their results, the gonosomes were recognised as early as spermatogonial prophase and prometaphase due to their precocious condensation, positive heteropycnosis, and association. However, our data showed the sex chromosomes were indistinguishable at mitotic prophase and prometaphase from autosomes. It was possible to recognise them from autosomes at the beginning of meiotic prophase only due to positive heteropycnosis.
In conclusion, our study described the karyotype features of five araneomorph spiders for the first time and confirms some findings of Král et al. 2011 for P. islamita.